Naproxen-based non-steroidal anti-inflammatory drug with low gastric toxicity

ABSTRACT

A new derivative of naproxen, 3-((S)-2-(6-methoxynaphth-2-yl) propanoyloxy)-4,5-bis (((S)-2-(6-methoxynaphth-2-yl) propanoyloxy)-methyl)-2-methylpyridinium (S)-2-(6-methoxynaphth-2-yl) propanoate has a high anti-inflammatory, analgesic and antipyretic activity, as well as low acute toxicity and gastrotoxicity, it can be used in the pharmaceutical industry, medicine and veterinary.

RELATED APPLICATIONS

This application is a Continuation application of InternationalApplication PCT/RU2017/000808, filed on Oct. 31, 2017, which in turnclaims priority to Russian Patent Application RU2016143071, filed Nov.2, 2016, both of which are incorporated herein by reference in theirentirety.

FIELD OF THE INVENTION

The invention relates to a new formula I naproxen derivative which has ahigh anti-inflammatory, analgesic and antipyretic activity, as well aslow acute toxicity and gastrotoxicity and which can be used in thepharmaceutical industry, medicine and veterinary medicine.

BACKGROUND OF THE INVENTION

The main indications for the prescription of non-steroidalanti-inflammatory drugs (hereinafter NSAIDs) are inflammatory processesof different nature and localization, pain and fever. NSAIDs are one ofthe most widely used groups of drugs. For example, NSAIDs are prescribedto about 20% of inpatients with various diseases of internal organs[Guidelines for Pre-clinical Drug Research. Part one/edited by A. N.Mironov.—M.: Grif and K Publ., 2012. p. 944].

The main element of the mechanism of action of NSAIDs is the suppressionof the synthesis of inflammatory mediators—prostaglandins. During theprocess of alteration (the first stage of inflammation) the cellmembrane releases phospholipids which under the action of the enzyme ofphospholipase A2 are metabolized to arachidonic acid. Arachidonic acid,in turn, is metabolized in two ways: cyclooxygenase (COX) andlipoxygenase (LOX). NSAIDs inhibit only COX, so they block thedevelopment of only the second stage of inflammation [Guidelines forPre-clinical Drug Research. Part one/edited by A. N. Mironov. —M.: Grifand K Publ., 2012. p. 944].

There are 2 COX isoenzymes: COX-1 (constitutional, normally existing)controls the production of prostanoids that regulate physiologicalfunctions of the stomach, vessels and kidneys; COX-2 (induced) isinvolved in the synthesis of prostaglandins in inflammation. COX-2 isnormally absent and is formed under the influence of tissue factors thatinduce an inflammatory response (cytokines, etc.). It is believed thatthe anti-inflammatory effect of NSAIDs is due to the inhibition ofCOX-2, and the side effects are due to the inhibition of COX-1[Guidelines for Pre-clinical Drug Research. Part one/edited by A. N.Mironov. —M.: Grif and K Publ., 2012. p. 944].

The most known NSAIDs are ketoprofen (3-benzoyl-alpha-methylbenzeneacetic acid), ibuprofen ((RS)-2-(4-isobutylphenyl) propionic acid),diclofenac (2-[(2,6-dichlorophenyl) amino] benzene acetic acid),indomethacin (1-(4-chlorobenzoyl)-5-methoxy-2-methyl-1H-indol-3-aceticacid) and naproxen ((S)-6-methoxy-α-methyl-2-naphthalenacetic acid).Currently naproxen occupies one of the leading positions in the group ofNSAIDs, because it has a longer effect than other NSAIDs and is welltolerated [Mashkovsky M. D. Lekarstvennye sredstva. [Medicinal Drugs.]16th ed., revised and modified—M.: Novaya Volna [New Wave] Publ., 2012.p. 1216].

The disadvantage of known to date NSAIDs, namely non-selectivecyclooxygenase inhibitors, is their pronounced gastric toxicity [Handa,O. The impact of non-steroidal anti-inflammatory drugs on the smallintestinal epithelium/O. Handa, Y. Naito, A. Fukui, T. Omatsu, T.Yoshikawa//J. Clin. Biochem. Nutr.—2014.—V. 54, N. 1.—P. 2-6.].Selective inhibitors of COX-2 are less gastrotoxic, but their negativeeffects on the cardiovascular system are known [Singh, B. K. Assessmentof nonsteroidal anti-inflammatory drug-induced cardiotoxicity/B. K.Singh, S. E. Hague, K. K. Pillai//Expert. Opin. Drug Metab.Toxicol.—2014.—V. 10, N. 2.—P. 143-156.]. Thus, on the date ofsubmission of the application materials, the problem of reducing theside effects of both types of NSAIDs—selective and non-selectivecyclooxygenase inhibitors—remains unresolved.

Thus, the development of safe and effective NSAIDs is one of the mostimportant tasks of pharmacotherapy and health care in general.

In the applicant view, based on the analysis of the level of technology,the most promising direction of development of anti-inflammatory drugsis search for non-selective NSAIDs. At the same time, as noted above,developers face the task of reducing side effects, the main of which isgastric toxicity.

Protection of carboxyl group of non-selective COX inhibitors is one ofthe main ways to reduce toxicity of this group of NSAIDs. Esterprotection is the most frequently used for this purpose [Liu, W.Synthesis and biological evaluation of curcumin derivatives containingNSAIDs for their anti-inflammatory activity [Text]/W. Liu, Y. Li, Y.Yue, et al.//Bioorg. Med. Chem. Lett.—2015.—V. 25, N. 15.—P.3044-3051.]. NSAIDs esters are typical promedications, which in thegastrointestinal tract (hereinafter referred to as the GIT) undergoenzymatic hydrolysis with gradual release of NSAIDs, providing aprolonged effect of the drug. In addition, promedications based onesters better penetrate cytoplasmic membranes of cells, thus having lessirritating effect on the mucous membrane of the GIT. Ascorbic acidesters with NSAIDs are known to be derivatives of arilacetic orarylpropionic acids, such as ibuprofen, ketoprofen, naproxen and theirsalts [EP 2431361, published on 21 Mar. 2012]. For the treatment ofarthritis, pain and inflammatory processes was proposed2-methansulfonatethyl ester of naproxen together with H2 receptorsantagonist [WO 200810106441, published 21 Aug. 2008]. This modificationof NSAIDs leads to a decrease in gastric toxicity, but at the same timesignificantly reduces the therapeutic effect. For this reason, thesecompounds have not been included in clinical practice.

The closest combination of coinciding features and achieved technicalresult to the claimed invention is the technical solution described inthe invention in the patent RU 2513089 “Non-steroidal anti-inflammatorydrugs based on pyridoxine derivatives”, the essence of which arepyridoxine derivatives of the general formula (I)

where:when R₂+R₃=—C(CH₃)₂— or —CH (CH₃)—;

when R₁=H; R₃=H;

having anti-inflammatory activity.

The prototype version containing three fragments of naproxen is aneffective anti-inflammatory compound. In particular, it has a pronouncedanti-inflammatory effect on the model of subacute (formalin) edema invivo.

However, on the model of acute (carrageenin) edema in vivo thisprototype was not effective enough. It is important to note that theprototype does not have at the same time a set of properties of theclaimed invention, namely high anti-inflammatory (on the model of acute(carrageenin) edema in vivo), analgesic and antipyretic activitycombined with low toxicity, including gastric toxicity. For this reason,the applicant does not consider this prototype as a comparison drug,instead of it a modern non-steroidal anti-inflammatory drug naproxen andits analogues are used for this purpose (table 3).

The claimed composition of the claimed invention differs from thatdescribed in the prototype by the presence of an additional fragment ofnaproxen, which, in the applicant's experimentally grounded view, causesthe appearance of pharmacologically significant positive effects. Thus,the combination in the claimed molecule of covalent ester bonds splitunder physiological conditions and non-covalent ionic bond linkingfragments of naproxen and pyridoxine, leads to the emergence ofsynergistic effects that are not obvious to a specialist. These effectsare expressed in increased anti-inflammatory, analgesic and antipyreticactivity, as well as in significantly reduced gastric toxicity and acutetoxicity. In particular, the claimed compound of formula I against thebackground of reduced gastric toxicity and acute toxicity ensures arapid onset of a pronounced anti-inflammatory effect on the model ofboth acute and subacute edema in vivo, whereby the applicant can resolvethe seemingly insurmountable problem typical of both the prototype andmany other NSAIDs including naproxen (the latter is also the prototypeof the claimed invention).

SUMMARY OF THE INVENTION

The objective of the claimed invention is to create a pharmaceuticaldrug based on naproxen, which has a high anti-inflammatory, analgesicand antipyretic activity in combination with low toxicity, includinggastric toxicity, significantly expanding the arsenal of known means ofthe specified destination.

The technical result of the invention is a new non-steroidalanti-inflammatory drug based on pyridoxine and naproxen, exhibiting highanti-inflammatory, analgesic and antipyretic activity on the backgroundof significantly lower toxicity, including gastric toxicity, comparedwith the known NSAIDs, including the prototype.

The problem is solved, and the claimed technical result is achieved bysynthesizing a compound of formula I and using it as ananti-inflammatory, analgesic and antipyretic agent with reduced gastrictoxicity:

The claimed invention is illustrated by the following materials:

Reaction sequence—chemical reaction sequence of synthesis of formula Icompound;

Table 1—Parameters of gradient elution mode at analytical HPLC;

Table 2—Acute toxicity of formula I compound in intragastricadministration;

Table 3—Comparative characteristics of formula I compound and some knownNSAIDs;

Table 4—Gastric toxicity of formula I compound with singleadministration at a dose of 2000 mg/kg;

Table 5—Effect of formula I compound on acute exudative inflammation inintragastric administration;

Table 6—Analgesic effect of formula I compound on rats;

Table 7—Antipyretic effect of formula I compound on rats.

Information confirming the composition and structure of the claimedcompound is shown in the examples of specific performance. The structureof the obtained compound was confirmed by ¹H and ¹³C NMR spectroscopy,UV spectroscopy, chromatography-mass spectrometry.

NMR spectra were recorded on the Bruker AVANCE-400 device. The chemicalshift was determined with respect to the signals of residual protons ofdeuterated solvents (¹H and ¹³C). Melt temperatures of products weredetermined using Stanford Research Systems MPA-100 OptiMelt at a heatingrate of 1° C./min. The UV spectrophotometer Evolution 300 (Thermoscientific) was used to study the spectral characteristics of thesolutions of the formula I compound. Specific rotation was determined onthe automatic polarimeter ADP440+(B&S) (England) using quartz matrixtube 100° Z. High-resolution mass spectra (HRMS) were recorded usingTripleTOF 5600, AB Sciex (Germany) mass spectrometer from methanolsolution by ionization—turboion spray (TIS)—at energy of impacts withnitrogen molecules equal to 10 eV.

Determination of purity of compound of formula I was performed byreverse phase HPLC using the values of the gradient elution mode ofTable 1, using a high performance liquid chromatograph LCMS-2010EV,Shimadzu (Japan) equipped with a XBridge C18 diode array detector (size4.6×50 mm, 3.5 μm). The temperature of the column thermostat is +40° C.,the flow rate is 1 ml/min, the volume of the introduced sample is 1mg/ml. UV detection was performed at an analytical wave length of 220nm, as well as 254 and 330 nm to determine impurities. As a moving phasewas used: channel A—0.1% by volume of formic acid in water, channelB—acetonitrile. The mode of operation of the pump is gradient elution,analysis time is 22 minutes.

TABLE 1 Gradient elution mode parameters for analytical HPLC Time, minVolume fraction of channel B component, % 0.01 5 10.00 70 25.00 0

EXAMPLES OF SPECIFIC IMPLEMENTATION OF THE CLAIMED TECHNICAL SOLUTION

Obtaining the claimed compound of formula I containing fragments ofpyridoxine (vitamin B₆) and naproxen is carried out in two stages usingcommercially available substrates, reagents and solvents according tothe reaction sequence below.

Figure Caption:

I—3-((S)-2-(6-methoxynaphth-2-yl) propanoiloxy)-4,5-bis(((S)-2-(6-methoxinaphtht-2-yl) propanoiloxy) methyl)-2-methylpyridinium(S)-2-(6-methoxynaphth-2-yl) propanoate

II—2-methyl-3-hydroxy-4,5-di-(hydroxymethyl) pyridine hydrochloride

III—(S)-2-(6-methoxy-2-naphthyl) propionic acid

IV—(2S,2'S)-(5-((S)-2-(6-methoxinaft-2-yl)propanoiloxy)-6-methylpyridine-3,4-diyl) bis (methylene) bis(2-(6-methoxynaphth-2-yl) propanoate)

Example 1. Obtaining (2S,2'S)-(5-((S)-2-(6-methoxinaphth-2-yl)propanoiloxy)-6-methylpyridine-3,4-diyl) bis (methylene) bis(2-(6-methoxinaphtht-2-yl) Propanoate) (Compound IV)

The round bottom flask is loaded with 28.35 g of pyridoxinehydrochloride II, 95.23 g of naproxene III, 50.52 g of 4-N,N-dimethylaminopyridine and 123.73 g of dicyclohexyl carbodiimide in 5 lof acetone. The reaction mixture is stirred until the formation of thedicyclohexyl urea precipitate is stopped, then the precipitate isfiltered, washed with 200 ml of acetone and the combined leachate isevaporated in a vacuum. The product is purified with columnchromatography on silica gel (eluent ethyl acetate-petroleum ether 1:1).Product IV is obtained in the form of white crystalline substance (yield82.0 g, 74%). In more detail, the preparation of this compound and itsproperties are described in the invention under the patent RU 2513098.

Example 2. Preparation of 3-((S)-2-(6-methoxinaphtht-2-yl)propanoiloxy)-4,5-bis (((S)-2-(6-methoxinaphtht-2-yl) propanoiloxy)methyl)-2-methylpyridinium (S)-2-(6-methoxinaphth-2-yl) Propanoate(Compound I)

Put 82.0 g of compound IV in a round bottom flask, add 1 l of acetoneand stir the mixture for 10 minutes. To the resulting solution add 18.22g of (S)-2-(6-methoxy-2-naphthyl) propionic acid (naproxene) III andstir until the precipitate is completely dissolved. Acetone is removedin vacuum on the rotary evaporator (90 rpm, residual pressure 10 mbar,temperature 40° C.). The precipitate is collected and dried under vacuumon a rotary evaporator (90 rpm, residual pressure 10 mbar, temperature80° C.). The yield of the claimed compound I is 100.1 g (99.9%).

Melting T is 145° C. [α]²⁴ _(D)=+22.8° (c=3.01, CH₂Cl₂). Absorptionspectra of solutions of formula I compounds are measured in acetonitrileand methylene chloride in the wave length range from 200 to 450 nm, inwhich case they have maximum absorption at 262, 272, 317 and 332 nm.Deviations of the position of the maxima are ±2 nm. Concentration ofsolutions is 40 μg/ml. The following signals are observed in the ¹H NMRspectrum (400 MHz, CDCl₃, δ, ppm, J/Hz): 1.33 (br d, 3H, CH₃, ³J=3.7);1.40 (d, 3H, CH₃, ³J=7.1); 1.50 (d, 6H, 2CH₃, ³J=7.1); 1.90 (br s, 3H,CH₃); 3.47 (br s, 1H); 3.65 (k, 1H, CH, ³J=7.1); 3.74-3.87 (m, 13H);4.50 (br s, 1 N); 4.80 (br s, 1 N); 4.95 and 5.04 (AB, 2H, CH₂,²J=−12.8); 6.95-7.66 (m, 20 N), 8.19 (s, 1H, CH). The following signalsare observed in the ¹³C NMR spectrum (100 MHz, CDCl₃, δ, ppm): 17.96;18.42; 18.52; 18.92; 45.02; 45.34; 45.38; 55.40; 57.13; 61.65; 105.66;105.73; 119.09; 119.13; 119.18; 119.38; 125.96; 126.09; 126.16; 126.20;126.38; 126.57; 127.30; 127.36; 127.49; 128.96; 129.01; 129.39; 129.64;133.78; 133.81; 133.86; 134.05; 134.13; 135.18; 135.21; 135.42; 136.16;144.78; 147.16; 152.75; 157.74; 157.79; 157.96; 171.95; 174.02; 179.30.

The target substance I is a salt of compound IV with naproxen. In thisregard, under the conditions of reversed-phase HPLC (table 1) it appearson the chromatogram as two peaks corresponding to compound IV andnaproxen, the mass spectra of which correspond to the calculated andliterature data (806.3324 and 229.0870, respectively).

The Study of the Acute Toxicity of Formula I Compound

The experiment was performed according to the fixed dose method inWistar rats of both sexes, 6 animals per group. The initial dose forintragastric administration was 5000 mg/kg. The solvent used was a 0.5%Tween-80 solution (Tween 80—polyoxyethylene, a derivative of sorbitanand oleic acid, a commercially available polymer), which was prepared bydissolving 0.5 g of Tween-80 in 100 ml of distilled water.

To prepare a dose of 5000 mg/kg 25 g of the test compound were weighedin polystyrene boats on scales Vibra (Shinko Denshi, Japan, cat. NoAF225DRCE), transferred to a 100 ml measuring flask with accuracy classA, adjusted to 100 ml with 0.5% water solution of Tween-80 and thesuspension of the preparation was stirred using a dispersantSilentCrusher (Heidolph, Germany, P/N 595-06000-00-3) to a homogeneousconsistency.

The introduction was carried out to animals deprived of food (for aperiod of not less than 8 hours) with free access to water. The volumeof administration was calculated individually for each animal, based onthe body mass recorded immediately before the introduction of thesubstance. Access to the feed was renewed an hour after theintroduction. Animals were observed individually after administrationfor 30 minutes, then at least once per hour for 4 hours, then daily oncea day for 14 days. Body mass was recorded immediately before theadministration of the preparation to calculate the volume ofadministration, then once every two days. If the animal died during thestudy, the time of death was determined and documented as accurately aspossible. The animal was weighed and dissected as soon as possible.Dying animals were weighed, euthanized and dissected. Animals wereeuthanized by inhalation of carbon dioxide. Toxic doses LD₁₀, LD₁₆, LD₅₀and LD₈₄ were calculated using probit analysis with IBM SPSS Statisticssoftware.

When administered intragastrically with a dose of 5000 mg/kg, a slightinhibition of the activity of the animal was observed. In 20-30 minutesthis symptom disappeared. After 1.5-2 hours, animals began to take foodand water. Three days later 1 rat (female) fell dead. Further throughoutthe study there were no mortality cases.

Throughout the experiment, all the main indicators of vital activity inexperimental animals corresponded to the norm and did not differ fromthe control ones. The animals had a good appetite, shiny coat, visiblemucous membranes were pale pink in color, the behavior corresponded tothis species of animal, no abnormalities were observed during theobservation period. The rats' body mass in 2 and 4 days after the startof the experiment increased by (1-4) % and (2-6) %, respectively, whichapproximately corresponds to the weight gain in the rats of the controlgroup. On the eighth day of the study the weight of animals increased by(2-8) %. At the end of the experiment, the weight increase was (5-13) %.Acute toxicity parameters are presented in table 2.

TABLE 2 Acute toxicity of formula I compound with intragastricadministration Number of animals in the group/ number of Animal deadspecies Sex Dose, mg/kg animals LD₁₀ LD₁₆ LD₅₀ LD₈₄ rat males 50006/0 >5000 >5000 >5000 >5000 females 5000 6/1 >5000 >5000 >5000 >5000

At the end of the experiment, euthanasia and pathomorphologicaldissection of control and experimental animals were performed. Nochanges were observed during the autopsy of rats. Corpses of animalswere of the correct constitution, with average or above average fatness.Natural openings: the mouth is closed, the tongue is in the mouth, themucous membrane of the lips and the gums are pale pink, smooth andshiny. Nasal openings—mucous membrane is pale pink, dry, no efflux,permeability is good. Ear shells are unchanged; the external auditorycanal is clean. Anus is closed, mucous membrane is pale pink. The hairis kept well, the fleece is shiny. The skin is elastic; the subcutaneousfiber is well expressed, has yellowish color and is elastic. The musclesare reddish, well developed, tendons and ligaments are white, elasticand durable. The configuration of bones and joints is not broken. Theposition of the organs of the thoracic and abdominal cavity isanatomically correct. There is no fluid in the thoracic and abdominalcavities. The patency of the pharynx and esophagus is not broken. Theheart is not changed in volume. The cavities of the heart contain asmall amount of non-clotted blood; the endocardium is smooth and shiny.The lungs are of pale pink color, evenly colored, with no signs ofswelling, the lobulation is well expressed. The spleen is not enlarged,with sharp edges, oblong in shape, has elastic consistency, red-brown incolor. The liver is not enlarged, with sharp edges, the shape is notchanged, the consistency is dense, and the color is red-brown. Thestomach contains a gray feed mass of a uniform consistency. The mucousmembrane of the stomach is pale gray. The mucous membrane of the thinand thick parts of the intestine is of pale pink or pale gray color. Thekidneys are bean-shaped, dark brown in color, in the paranephric bodythere is a moderate amount of fat, the capsule is easily separated, andthe boundary between the cortical and brain zones is expressed. Theurinary bladder is empty or filled up with urine of light yellow color,the mucous membrane is of pale pink color. Genitals are withoutabnormalities. Males' testicles are of elastic consistency, are in thecavity of the scrotum, have an elliptical shape. The females have normalovaries and uterus. The brain is not edematic; the brain matter iselastic, without hemorrhages.

Thus, the conducted studies showed that the claimed compound of theformula I after intragastric administration belongs to the 4th hazardclass, i.e. to low-hazard substances (GOST 12.1.007-76 “Harmfulsubstances. Classification and general safety requirements”) and itssafety in terms of LD₅₀ lethal dose exceeds most NSAIDs known to date(Table 3). For example, the claimed compound is 8 times less toxic thannaproxen.

TABLE 3 Comparative characteristics of the compound of formula I andsome known NSAIDs * ED₅₀ UD₅₀/ LD₅₀/ (mg/kg) ED₅₀ ED₅₀ acute UD₅₀ LD₅₀(safety (therapeutic The drug inflammation (mg/kg) (mg/kg) index) index)Compound of 19 >2000 >5000 >105 >263 formula I Ibuprofen 48 310 750 6.4516 Diclofenac 8 48 370 6 46 sodium Naproxen 15 49 620 3.2 42 Pyroxicam20 36 290 1.8 15 Phenylbutazone 56 120 430 2.1 7.7 Acetylsalicylic 98240 1600 2.45 16 acid Indomethacin 10 10 47 1 4.7 * Guidelines forPre-clinical Drug Research. Part one/edited by A. N. Mironov. - M.: Grifand K Publ., 2012. p. 944.

Gastric Toxicity of Formula I Compound

In the study of gastric toxicity were taken female and male Wistar ratsaged 6-7 weeks, with a body mass of 180-220 g. The number of animals ineach group was 10.

The compound of formula I was administered once intragastrically to ratsdeprived of food for 16 hours before the study. Three hours after theadministration of the suspension of the test substance at a dose of 2000mg/kg (in 0.5% aqueous solution of Tween-80), the animals wereeuthanized; their stomachs were removed, dissected along the lessercurvature, and washed in physiological solution to remove the contents.

Evaluation of gastric toxicity was performed on a 4-point scale:

0—no damage;

0.5—hyperemia;

1—single minor injuries (1 or 2 point hemorrhages);

2—multiple injuries (erosion, spot hemorrhages);

3—significant and multiple injuries of the mucosa (erosion, hemorrhage);

4—gross injuries covering the entire surface of the mucosa (massivehemorrhages, erosion, perforations).

According to the results of the assessment, UD₅₀ was determined the doseof the tested substance causing gastrotoxic (ulcerogenic) effectcorresponding to 2 points.

To prepare a dose of 2000 mg/kg, 32.0 g of the formula 1 compound wereweighed in polystyrene boats on scales Vibra (Shinko Denshi, Japan, cat.No. AF225DRCE), transferred to a 400 ml measuring flask with accuracyclass A, adjusted to 400 ml with 0.5% water solution of Tween-80 and thesuspension of the preparation was stirred using a dispersantSilentCrusher (Heidolph, Germany, P/N 595-06000-00-3) to a homogeneousconsistency. The introduction to rats is carried out in the amount of nomore than 5 ml/200 g.

Animals were euthanized by carbon dioxide inhalation method.

At a single introduction of formula I compound at a dose of 2000 mg/kg,the gastrotoxic effect corresponded to an average of 0.6 points in malerats and 0.4 in females (Table 4).

TABLE 4 Gastric toxicity of formula I compound with singleadministration at a dose of 2000 mg/kg; Gastrotoxic effect (averaged),Number of Dose, points UD₅₀ (mg/kg) Group animals mg/kg males femalesmales females Compound of 10/10 2000 0.6 0.4 >2000 >2000 formula I

Thus, for a compound of formula I, the UD₅₀ value is >2000 mg/kg, whichexceeds the similar parameter for most NSAIDs (Table 3). For example,the claimed compound is 40 times less toxic than naproxen.

Anti-Inflammatory Activity of the Compound of Formula I In Vitro

The structural enzyme cyclooxygenase 1 (COX 1) is expressed in variouscell types and is involved in ensuring their normal (physiological)functional activity. Cyclooxygenase 2 (COX 2) is responsible for thesynthesis of prostaglandins in severe inflammation conditions.

The analysis of COX activity consisted in measuring the peroxidaseactivity of cyclooxygenase. During the reaction of PGG2 (prostaglandinG2) with ADHP (10-acetyl-3,7-dihydrofenoxazine), presented in the kitused (“Cyclooxygenase (COX) Activity Assay Kit (Fluorometric)”(Biovision, cat # K 549-100) as a COX probe, a fluorescent componentresorufin is formed, the absorption wavelength of which is 530-540 nm,the emission wavelength is (585-595) nm. The fluorescence intensity isdirectly proportional to the residual activity of COX in the sample.Skin fibroblast cells (HSF) were selected as a source of cyclooxygenaseenzyme. The fibroblast cell lysate was shown to have a COX activity of2.131 μU/mg.

Preparation of Cell Lysate.

The cells of the skin fibroblasts (2-6×10⁶) are washed once with 10 mlof phosphate buffer. The cells are resuspended in 5 ml of buffer andtransferred to a 15 ml tube. They are centrifuged at 1500 g for 4minutes. The supernatant is then poured, the cell pellet is resuspendedin 0.5-1 ml of cold lysing buffer with a protease cocktail(approximately 0.4 ml of buffer per 100 μl of cell pellet), andcentrifuged at 4° C. for 15 minutes at 10,000 g. The supernatant isseparated and used as a source of COX.

Preparation of Reagents.

COX cofactors are dissolved 200 times by adding 2 μl of cofactors to 398μl of buffer immediately before use. 65 μl of arachidonic acid and 65 μlof NaOH are mired, and then diluted 10 times with 1170 μl of distilledwater. The solution is stable for 1 hour.

The reaction mixture is prepared (for 2 parallel wells) by mixing thefollowing reagents indicated below:

-   -   COX probe—2 μl;    -   dissolved cofactor—4 μl (stable for 1 hour);    -   cell lysate—20 μl;    -   with the help of COX buffer the volume is brought to 172 μl.

Preparation of the Tested Substances:

Naproxen stock solutions and substances of the formula I in dimethylsulfoxide (DMSO) with a concentration of 1000 μM are used to preparepreliminary dilutions in 2-step increments. Respectively, 5concentrations from 62.5 to 1000 μM are obtained.

Using a multichannel pipette, reagents are added to the appropriateplate wells: first, the reaction mixture, then the solutions of the testsubstances in DMSO in a series of concentrations and control substances(inhibitor COX 1 SC560 and inhibitor COX 2 Celecoxib), pure solvent DMSO(for wells with higher activity). Two replications are used. They areincubated for 5 minutes.

Initiating a Reaction.

Before the start of the reaction, a single fluorescence measurement isperformed in all wells of the plate to account for the own signal of thetest and control substances. Then 10 μl of arachidonic acid solution isadded to all wells of the plate using a multichannel pipette to initiatethe reaction. After adding arachidonic acid, the fluorescence(Ex/Em=535/587 nm) is immediately measured in kinetic mode every 15seconds for 30 minutes.

The fluorescence parameter of images with control and tested substancesis calculated according to the formula:Average Net RFU=Average RFU−Average Blank RFU

The percentage of COX inhibition is calculated according to the formula:

$\left( {1 - \frac{{Average}\mspace{14mu}{Net}\mspace{14mu}{Inhibitor}\mspace{14mu}{RFU}}{{Average}\mspace{14mu}{Net}\mspace{14mu}{RFU}\mspace{14mu}{for}\mspace{14mu}{non}\text{-}{inhibited}}} \right) \times 100$

Further, using the available data points for the test substance and thereference substance (naproxen), we construct the most suitable sigmoidinhibition curve. Based on this curve, the IC₅₀ of the test sample forCOX is calculated.

According to the recommendations of the manufacturer of the kit used,the COX-1 inhibitor SC560 and the COX-2 inhibitor celecoxib are added tothe reaction mixture in a volume of 2 μl (this amount is sufficient tocompletely inhibit the appropriate isoform). The residual activity ofCOX contained in cell lysate after treatment by SC560 and celecoxib is72.8% and 32.0% respectively.

In the study of inhibitory activity, the concentration of naproxen andthe tested substance in the well is from 1.25 to 20 μM.

To determine the proportion of inhibition by the preparation of each ofthe isoforms, the studied substances are tested in a concentrationpattern in the presence of a selective inhibitor COX 1 SC560, as well asin the presence of a selective inhibitor COX 2 celecoxib. The inhibitoris added in sufficient quantities to completely inhibit thecorresponding isoform of the enzyme, while the other isoform remainsactive.

Conventionally assuming the activity in the presence of only the testpreparation as 100%, the residual activity of COX 1 and COX 2 and thepercentage of inhibition of each isoform by the preparation arecalculated. The compound of formula I exhibits preferential inhibitoryactivity against the isoform of COX-2.

Thus, the naproxen IC₅₀ (prototype) is 9.56 μM, which is 1.25 times theIC₅₀ of the compound of formula I, equal to 7.7 μM. From the above, itcan be concluded that the claimed compound of formula I is moreeffective as a cyclooxygenase inhibitor, since it acts at a lowerconcentration.

Anti-Inflammatory Activity of the Compound of Formula I In Vivo

The experiment was carried out using female and male Wistar rats aged6-7 weeks with a body mass of 180-220 g. The number of animals in eachgroup was 10.

The acute inflammatory reaction (edema) was reproduced by subplantar(under plantar or plantar aponeurosis) administration of 0.1 ml of 1%carrageenin solution (sulfated polysaccharide from Irish sea moss). Theseverity of the inflammatory reaction was assessed 3 hours after theinduction of inflammation by a change in the volume of the paw(oncometric). The substance of formula I was administered into thestomach with the probe 1 hour before the introduction of the 1% solutionof carrageenin. A 0.5% Tween-80 solution was used as a negative control.The anti-inflammatory effect, assessed by a decrease in edema, wasexpressed as a percentage of the control. According to the results ofthe action of four doses of the analyte, the calculation of ED₅₀ wasperformed.

To prepare a dose of 50 mg/kg, 1000 mg of the formula I compound wereweighed in polystyrene boats on scales Vibra (Shinko Denshi, Japan, cat.No AF225DRCE), transferred to a 200 ml measuring flask with accuracyclass A, adjusted to 200 ml with 0.5% water solution of Tween-80 and thesuspension of the preparation was stirred using a dispersantSilentCrusher (Heidolph, Germany, P/N 595-06000-00-3) to a homogeneousconsistency. The introduction to rats was carried out in the amount ofno more than 1 ml/100 g. The concentration of the resulting solution was5 mg/ml.

To prepare a dose of 20 mg/kg, 40 ml of solution with a concentration of5 mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% water solution ofTween-80 and the suspension of the preparation was stirred using adispersant to a homogeneous consistency.

To prepare a dose of 10 mg/kg, 20 ml of solution with a concentration of5 mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% aqueous solution ofTween-80 and the suspension of the preparation was stirred with amagnetic stirrer MR Hei-Standard (Heidolph, Germany) to a homogeneousconsistency.

To prepare a dose of 5 mg/kg, 4 ml of solution with a concentration of 5mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% water solution ofTween-80 and the suspension of the preparation was stirred with amagnetic stirrer to a homogeneous consistency.

Animals were observed individually after administration for 30 minutes,then at least once an hour for 4 hours. Body mass was recorded justbefore the administration of the preparation to calculate the volume ofadministration.

Animals were euthanized by inhalation of carbon dioxide.

When a dose of 50, 20, 10 and 5 mg/kg of the compound of formula I wasadministered to male rats, inflammatory edema decreased by 62.34, 51.25,23.57 and 12.39%, respectively, in contrast to the control group. Infemale rats, with a dose of 50, 20, 10 and 5 mg/kg of the compound offormula I administered, the edema decreased by 65.47, 67.89, 38.64 and13.37%, respectively. The results are presented in table 5.

TABLE 5 Effect of the compound of formula I on acute exudativeinflammation after intragastric administration Anti-inflam- Dose, Pawvolume matory Group mg/kg 0 3 h effect, % Male rats Compound 50 mg/kg1.04 ± 0.20 1.54 ± 0.45 37.66 ± 19.16 of 20 mg/kg 0.93 ± 0.05 1.51 ±0.16 48.75 ± 16.39 formula I 10 mg/kg 0.93 ± 0.06 1.84 ± 0.23 76.43 ±22.74  5 mg/kg 0.89 ± 0.07 1.87 ± 0.21 87.61 ± 19.75 Control — 0.98 ±0.09 2.23 ± 0.31 100.00 ± 16.92  (Tween-80) Female rats Compound 50mg/kg 0.91 ± 0.05 1.26 ± 0.23 34.53 ± 18.52 of 20 mg/kg 0.91 ± 0.09 1.23± 0.20 32.11 ± 15.06 formula I 10 mg/kg 0.96 ± 0.06 1.60 ± 0.16 61.36 ±17.12  5 mg/kg 1.00 ± 0.03 1.94 ± 0.20 86.63 ± 17.71 Control — 0.86 ±0.10 1.80 ± 0.28 100.00 ± 19.39  (Tween-80)

According to the results of statistical processing of the obtained datathe ED₅₀ of the compound of formula I on males is 23 mg/kg, on femalesis 14 mg/kg. The mean value of ED₅₀ without sex distinction is 18.5(˜19) mg/kg.

According to the literature, ED₅₀ of naproxen is 15 mg/kg, ibuprofen—48mg/kg, diclofenac sodium—8 mg/kg, pyroxicam—20 mg/kg, phenylbutazone—56mg/kg, indomethacin −10 mg/kg, acetylsalicylic acid—98 mg/kg [Guidelinesfor Pre-clinical Drug Research. Part one/edited by A. N. Mironov. —M.:Grif and K Publ., 2012. p. 944]. Thus, the applicant experimentallyproved the presence of a high anti-inflammatory activity of a compoundof formula I, which is superior in its value (in four cases out ofseven) or comparable to the anti-inflammatory activity of modern NSAIDs.

Analgesic Effect of Formula I Compound

In the study of analgesic properties of the formula I compound, weretaken female and male Wistar rats aged 6-7 weeks with a body mass of180-220 g. The number of animals in each group was 10.

Chronic immune inflammation was modeled in rats by subplantaradministration of 0.1 ml of a 1% solution of carrageenin (a sulfatedpolysaccharide from Irish sea moss) into the right hind paw. Thesubstance was administered intragastrically using a gastric probe.Naproxen was used as a comparison drug, 0.5% Tween-80 solution was usedas a negative control. The anti-inflammatory effect, assessed by adecrease in edema, was expressed as a percentage relative to thecontrol; according to the results of the action of four doses of thetest substance, the calculation of ED₅₀ was performed.

Inflammatory hyperalgesia (increased pain sensitivity of inflamedtissues in rats) was caused by carrageenin and was assessed by decreaseof the pain sensitivity threshold—PST (by PST difference) to mechanicalirritation (squeezing) of the paw tissue of the animal before theintroduction of carrageenin and 3 hours after it. The measurement wasperformed on the inflamed paw. Used analgesimeter Ugo Basile S.R.L.(Italy) provides a gradual increased afterload on the rat's inflamed pawuntil a pain reaction appears (assessed by squeaking of animal orwithdrawing the paw). The analyte was administered 2 h after carrageeninadministration. The analgesic effect with evaluation of ED₅₀ wasassessed by a decrease in hyperalgesia 1 h after intragastricadministration of the analyte. An increase in the pain responsethreshold under the influence of the substances under study wasexpressed in an increase in the force of compression of the limb andcharacterized the intensity of the analgesic effect of the preparation.After the experiment, animals were euthanized by carbon dioxideinhalation method.

To prepare a dose of 50 mg/kg, 1000 mg of the formula I compound wereweighed in polystyrene boats on scales Vibra (Shinko Denshi, Japan, cat.No AF225DRCE), transferred to a 200 ml measuring flask with accuracyclass A, adjusted to 200 ml with 0.5% water solution of Tween-80 and thesuspension was dispersed using a dispersant SilentCrusher (Heidolph,Germany, P/N 595-06000-00-3) to a homogeneous consistency. Theintroduction to rats was carried out in the amount of no more than 1ml/100 g. The concentration of the resulting solution was 5 mg/ml.

To prepare a dose of 20 mg/kg, 40 ml of solution with a concentration of5 mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% water solution ofTween-80 and the suspension was dispersed to a homogeneous consistencyusing a dispersant.

To prepare a dose of 10 mg/kg, 20 ml of solution with a concentration of5 mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% aqueous solution ofTween-80 and the suspension was stirred to a homogeneous consistencywith a magnetic stirrer MR Hei-Standard (Heidolph, Germany).

To prepare a dose of 5 mg/kg, 4 ml of solution with a concentration of 5mg/ml (50 mg/kg) were taken, transferred to a 100 ml measuring flaskwith accuracy class A, adjusted to 100 ml with 0.5% water solution ofTween-80 and the suspension was stirred to a homogeneous consistencywith a magnetic stirrer.

The results of the experiment are presented in table 6.

TABLE 6 Analgesic effect of compound of formula I on rats Dose, Pawvolume Analgesic effect, total/ Group Sex mg/kg 0 hours 3 hoursAnalgesic effect, % analgesic effect Compound males 50 3.4 ± 2.3 10.6 ±6.3  367.9 ± 220.1 10/8 of formula I females 8.9 ± 5.7 12.4 ± 6.5  484.4± 251.8 10/7 males 20 6.4 ± 5.7 12.0 ± 6.2  418.1 ± 215.7 10/9 females4.6 ± 3.3 9.1 ± 7.4 353.5 ± 289.7 10/9 males 10 4.2 ± 2.4 12.0 ± 4.6 453.7 ± 159.9 10/10 females 4.3 ± 2.2 10.5 ± 7.2  408.2 ± 279.3 10/8males 5 4.4 ± 2.7 5.5 ± 2.5 259.9 ± 229.7 10/7 females 3.9 ± 1.4 7.2 ±3.2 282.0 ± 125.9 10/9 Control males 0 5.7 ± 2.0 2.9 ± 0.9 100.0 ± 30.010/10 females 4.7 ± 1.5 2.6 ± 1.4 100.0 ± 52.6 10/9 Naproxen males 155.0 ± 2.9 6.7 ± 4.6 234.5 ± 160.2 10/7 females 5.0 ± 6.2 5.0 ± 3.2 195.3± 125.3 10/7

From the presented results, it follows that the claimed compound offormula I exhibits a pronounced anesthetic effect already at a dose of 5mg/kg, while naproxen (prototype) has a similar effect only at a dose of15 mg/kg, which is a demonstrative fact of its higher efficiency. Thus,the therapeutic index (LD₅₀/ED₅₀) of the claimed compound in terms ofits analgesic activity is more than 1000 against 42 for naproxen, andthe safety index (UD₅₀/ED₅₀) is more than 400 against 3.2 for naproxen.

Antipyretic Effect of Compound of Formula I on Rats

In the study of antipyretic properties of the formula I compound, weretaken female and male Wistar rats aged 6-7 weeks, with a body mass of180-220 g. The number of animals in each group was 10.

The febrile reaction was caused by subcutaneous administration of a 20%suspension of baking yeast. The rectal temperature was measured with anelectrothermometer prior to the introduction of yeast and 18 hours afterit (the difference of these measurements is the estimated hyperthermicreaction). The preparation was administered to animals at the peak ofhyperthermia once (after 18 hours). The antipyretic effect was evaluatedby hyperthermia decrease 2 hours after the injection of the testsubstance, and then the effect dynamics was recorded at hourly intervalsfor 7 hours.

To prepare a dose of 75 mg/kg, 1500 mg of the formula I compound wereweighed in polystyrene boats on scales Vibra (Shinko Denshi, Japan, cat.No AF225DRCE), transferred to a 200 ml measuring flask with accuracyclass A, adjusted to 200 ml with 0.5% water solution of Tween-80 and thesuspension of the preparation was stirred to a homogeneous consistencyusing a dispersant SilentCrusher M (Heidolph, Germany). The introductionto rats was carried out in the amount of no more than 1 ml/100 g. Theconcentration of the resulting solution was 7.5 mg/ml.

To prepare a dose of 50 mg/kg, 66.7 ml of solution with a concentrationof 7.5 mg/ml (75 mg/kg) were taken, transferred to a 100 ml measuringflask with accuracy class A, adjusted to 100 ml with 0.5% water solutionof Tween-80 and the suspension of the preparation was stirred to ahomogeneous consistency using a dispersant.

To prepare a dose of 20 mg/kg, 26.7 ml of solution with a concentrationof 7.5 mg/ml (75 mg/kg) were taken, transferred to a 100 ml measuringflask with accuracy class A, adjusted to 100 ml with 0.5% aqueoussolution of Tween-80 and the suspension of the preparation was stirredto a homogeneous consistency with a magnetic stirrer MR Hei-Standard(Heidolph, Germany). The difference in temperature after preparationadministration and after 18 hours of induction with yeast was taken asthe parameter of hyperthermia decrease.

Animals were observed individually after administration throughout thestudy. Body mass was recorded just before the administration of thepreparation to calculate the volume of administration. Descriptivestatistics were used for all data: there were calculated the averagevalue and the standard error of the mean, results were represented inthe final table 7. Wilcoxon nonparametric criterion was used forstatistical comparison of experimental groups. Differences weredetermined at the 0.05 level of significance. The statistical analysiswas carried out using the R-studio program. The results of the study ofantipyretic effects of various doses of formula I compound are presentedin table 7.

TABLE 7 The antipyretic activity of the formula I compound Δ t after Δ tafter Δ t after Δ t after Δ t after Δ t after 2 hours, 3 hours, 4 hours,5 hours, 6 hours, 7 hours, Dose, Sex of (M ± (M ± (M ± (M ± (M ± (M ±Group mg/kg animals SD) SD) SD) SD) SD) SD) Compound 20 Males 0.13 ±0.31  0.24 ± 0.37  0.02 ± 0.25  0.1 ± 0.26 −0.21 ± 0.22  0.04 ± 0.28  offormula I Females −0.07 ± 0.17  −0.27 ± 0.17  −0.35 ± 0.16  0.34 ± 0.18 −0.01 ± 0.22  −0.08 ± 0.19  50 Males 0.22 ± 0.16  0.36 ± 0.17* 0.39 ±0.11* 0.25 ± 0.2  0.11 ± 0.19  0.09 ± 0.18  Females 0.87 ± 0.09* 0.91 ±0.14* 0.66 ± 0.12* 0.53 ± 0.14* 0.61 ± 0.22*  0.5 ± 0.15* 75 Males 0.76± 0.15* 1.15 ± 0.18* 1.38 ± 0.14* 1.31 ± 0.25* 0.96 ± 0.16* 0.92 ± 0.19*Females 0.97 ± 0.19* 0.96 ± 0.15* 1.39 ± 0.09* 1.52 ± 0.22* 1.51 ± 0.17*1.17 ± 0.2*  Naproxen 15 Males 1.39 ± 0.25* 1.49 ± 0.21*  1.6 ± 0.19*1.51 ± 0.25* 1.53 ± 0.19* 1.44 ± 0.24* 15 Females 1.64 ± 0.23* 1.35 ±0.15* 1.58 ± 0.21* 1.21 ± 0.18* 1.09 ± 0.25* 0.74 ± 0.14* Control —Males 0.08 ± 0.18  −0.49 ± 0.13  −0.46 ± 0.2   −0.68 ± 0.16  −0.28 ±0.2   0.19 ± 0.21  0.5% Tween — Females −0.31 ± 0.14  −0.36 ± 0.16 −0.42 ± 0.18  −0.65 ± 0.2   −0.25 ± 0.18  −0.47 ± 0.21  80 *p < 0.05 incomparison with naproxen

18 hours after the subcutaneous introduction of baking yeast suspensiona positive increase in rectal temperature was observed in groups ofanimals on average by 1.5° C. No decrease in temperature was observed inthe control group of animals during the entire observation period (up to7 hours after preparation administration). With the introduction of theformula I compound to both males and females at a dose of 20 mg/kg noantipyretic action was observed. When the dose was increased to 50mg/kg, a distinct decrease in temperature was observed in the group offemales—so, 2 and 3 hours after the substance was introduced, thetemperature was lower than before the introduction by 0.9° C., in 4, 5,6 and 7 hours after the introduction the decrease was (0.5-0.7) ° C.

With the introduction of formula I compound at a dose of 50 mg/kg, asignificant decrease in temperature was observed in 3-4 hours afteradministration. The level of decrease was equal to (0.34-0.97) ° C.

The greatest effect was observed in animal groups which wereadministered formula I compound at a dose of 75 mg/kg. Thus, with theintroduction of the substance at a dose of 75 mg/kg throughout theexperiment, the temperature decreased significantly and was less thanbefore the introduction for (0.76-1.5) ° C.

With the introduction of naproxen at a dose of 15 mg/kg (or 65 μmol/kg),the temperature also decreased significantly. The level of temperaturedecrease was (0.74-1.6) ° C.

Summarizing the results obtained, it should be concluded that, at a doseof 75 mg/kg, the claimed compound has antipyretic properties at thelevel of naproxen dose of 15 mg/kg. It should be particularly noted thatin molar terms these doses do not practically differ, since the molarmass of naproxen is significantly lower than the molar mass of thecompound of formula I, and is equal to 230 g/mol versus 1036 g/mol ofthe claimed compound. The therapeutic index (LD₅₀/ED₅₀) of the claimedcompound in terms of its antipyretic activity is more than 67 against 42for naproxen, and the safety index (UD₅₀/ED₅₀) is more than 27 against3.2 for naproxen.

Thus, it follows from the above that the applicant obtained a newcompound based on pyridoxine andnaproxen-3-((S)-2-(6-methoxynaphth-2-yl) propanoyloxy)-4,5-bis(((S)-2-(6-methoxynaphth-2-yl)-propanoyloxy) methyl)-2-methyl-pyridinium(S)-2-(6-methoxynaphth-2-yl)-propanoate, which simultaneously has highanti-inflammatory, analgesic and antipyretic properties that aresuperior or comparable with similar properties of naproxen (prototype),while maintaining extremely low toxicity (including gastric toxicity).

In general, the claimed compound is 8 times less toxic and 40 times lessgastrotoxic compared to naproxen (prototype).

The claimed invention meets the criterion of “novelty” applied toinventions, because from the studied level of technology, no technicalsolutions were found that coincide with the claimed one on essentialfeatures leading to the realization of the stated technical results,which are new pyridoxine and naproxen derivatives having low toxicity,including gastric toxicity. The claimed chemical compound expands therange of products for the treatment of rheumatic diseases, as well asdiseases accompanied by inflammation, pain and fever, and isfundamentally new, unparalleled in the world in terms of itseffectiveness and safety of use.

The claimed invention meets the criterion of “inventive step” applied toinventions, because it is not obvious to specialists in this field oftechnology, namely, from the studied level of technology noanti-inflammatory, analgesic and antipyretic drugs of the claimedstructure were identified.

The claimed invention meets the criterion of “industrial applicability”,as it can be implemented at any specialized enterprise using standardequipment, well-known domestic materials and technologies. The applicantobtained in the laboratory the target product—non-steroidalanti-inflammatory drug based on naproxen, with the claimed technicalresults and achieved all the claimed objectives.

What is claimed is:
 1. A non-steroidal anti-inflammatory drug based on naproxen formula (I):

having anti-inflammatory, analgesic and antipyretic activity, as well as reduced acute toxicity and gastric toxicity. 